The separation, manipulation and sorting of biological components is essential to the success of microfluidic and other portable diagnostics. Current needs in the areas of low cost battlefield diagnosis and homeland security requires robust and inexpensive platforms that combine sample processing, detection, and signal transduction into an integrated package. These diagnostic sensors must satisfy three major criteria: low power, little skills training for the end-user and the ability to directly sample “dirty” environments or complex matrices.
Sample processing of complex matrices, however, remains the limiting factor in any portable device. An onboard process must be established that ensures both the sample purity and sample concentration are high enough to guarantee detection success. Current “macroscopic” or laboratory solutions, including electrokinetics, flow cytometry, centrifuging, and hydrodynamic sorting are not suitable for portable devices. Furthermore “microscopic” solutions including magnetic activated or fluorescent activated cell sorting require relatively pure samples and a hybridization step, which limits throughput capacity and response time.
Membrane Bioreactors enrich too many cell types and are prone to biofouling. Membranes provide environmental engineers with the ability to prevent the removal of all microbial cells. While this approach is advantageous because it allows for the enrichment of slowly growing microbes with specialized metabolic functions (ex. Methanogens), these systems suffer from three problems:
1) Enrichment of contaminants, such as filamentous bacteria that can cause foaming of the biosolids. This foaming can result in loss of biosolids with subsequent poor performance.
2) Enrichment of all cell types results in high levels of biomass, which is difficult to mix and/or aerate.
3) The membranes are also prone to biofouling which require higher pressures to move liquid across the membrane. Membranes are typically cleaned with harsh chemicals or simply replaced.
Before the 1970's the phylogeny of the Prokaryotes was based on crude comparisons of morphology and pattern of substrate utilization and was largely ignored due to the presumed simplicity of the organisms. Carl Woese used a different strategy to tackle Prokaryotic phylogeny. He focused on sequence comparisons of the ribosome, which is a biomolecule found in all life forms. The ribosome is an essential macromolecule that is involved in the translation of messenger RNA into proteins. Woese argued that since protein synthesis is an essential function for life, the ribosome could not withstand major sequence changes or life would cease. He then targeted one molecule, the 16S rRNA of Prokaryotes and the analogous 18S rRNA for Eukaryotes, and did comparisons by sequence analysis [1]. A new phylogeny of all life was discovered and to his surprise (and other biologists), the old phylogeny of Eukaryotes and Prokaryotes was discarded for a three-kingdom version that included Bacteria, Archaea, and Eucarya.
Over time, most biologists have accepted this paradigm shift. To date, 35 Bacteria phyla and 18 Archaea phyla were identified, despite only having 30 cultivatable representatives for both [2]. The branch lengths of the major Bacteria groups suggest significant differences in the genetic makeup and phenotypic characteristics. For example, the Gram positive bacteria form a distinctive phylogenetic group and have a distinctly different cell wall compared to the rest of the bacteria, which are Gram negative. Each of these major groups of Bacteria has gross differences in their surface properties that can be exploited by the present invention.